The Invention disclosed herein relates to calcium phosphate (CaP) microcarriers and microspheres and their use, for example, in cell culturing systems, chromatography analysis and processing, and implantable materials useful for biomedical implants.
Revolutionary advances in biotechnology and genetic engineering have created enormous potential for marketing cellular by-products, including for example, proteins, including protein pharmaceuticals such as interferon, monoclonal antibodies, TPA (Tissue Plasminogen Activator), growth factors, insulin, and cells for transplantation. The demand for these products has grown tremendously and will continue to do so, creating a need for efficient methods of producing industrial quantities of cell-derived pharmaceuticals and other products. Further, the demand for efficient methods of analyzing and isolating biological products through chromatographic technology, and the need to improve bio-implantables continues to grow.
Research and study of cell structure and morphology are fundamental to continued progress in the diagnosis and treatment of human diseases. Numerous cell products are of vital importance therapeutically and commercially, including, for example, hormones, enzymes, viral products, vaccines, and nucleic acids. The production of these products requires large scale cell culture systems for their production.
Mammalian cells can be grown and maintained in vitro, but are generally anchorage-dependent, i.e., they require a solid surface or substrate for growth. The solid substrate is covered by or immersed in a nutrient medium particular to the cell type to be cultured. The nutrient medium and solid substrates are contained in a vessel and provided with an adequate supply of oxygen and carbon dioxide to support cell growth and maintenance. Cell cultures may be batch systems, in which nutrients are not replenished during cultivation although oxygen is added as required; fed batch systems, in which nutrient and oxygen are monitored and replenished as necessary; and perfusion systems, in which nutrient and waste products are monitored and controlled (Lubiniecki, Large Scale Mammalian Cell Culture Technology, Marcel Dekker, Inc., New York, 1990).
The primary commercial systems used for mammalian cell culture use solid matrix perfusion and microcarrier bead systems (Lubineicke, supra). The solid matrix perfusion systems utilize glass columns packed with glass beads or helices, which form a matrix as the solid substrate for cell growth. Once cells have attached to the matrix, medium is continuously recycled from a storage vessel for support of cell growth and maintenance. A similar perfusion system uses hollow fibers as the solid matrix instead of beads.
In microcarrier systems, small spheres are fabricated, for example, from an ion exchange gel, dextran, polystyrene, polyacrylamide, or collagen-based material. These materials have been selected for compatibility with cells, durability to agitation and specific gravities that will maintain suspension of the microcarriers in growth mediums. Microcarriers are generally kept in suspension in a growth medium by gently stirring them in a vessel. Microcarrier systems are currently regarded as the most suitable systems for large-scale cell culture because they have the highest surface to volume ratio and enable better monitoring and control. Nevertheless, current microcarrier culture systems have a number of serious disadvantages: small microcarrier cultures cannot be used to inoculate larger microcarrier cultures; therefore, a production facility must use other culture systems for this purpose; the cost of microcarriers is high, which can necessitate reprocessing of the microcarriers for reuse with the attendant costs; and the oxygen transfer characteristics of existing microcarrier systems are rather poor.
Specific forms of calcium phosphate ceramic have been identified for use in microcarriers to support anchorage-dependent cells in suspension. These specialized ceramics provide a material which is biomimetic, i.e., it is composed of mineral species found in mammalian tissues, and which can be further applied to a variety of in vitro biological applications of commercial interest. A number of common cell lines used in industrial applications require attachment in order to propagate and need substrate materials such as microcarriers for large scale cultivation. U.S. Pat. No. 4,757,017 (Herman Cheung) teaches the use of solid substrates of mitogenic calcium compounds, such as hydroxylapatite (HA) and tricalcium phosphate (TCP) for use in in vitro cell culture systems for anchorage-dependent mammalian cells. The unique features of this technology include the growth of cells in layers many cells thick, growth of cells that maintain their phenotype and the ability to culture cells for extended periods of time. Cheung demonstrated the application of this technology for culturing red blood cells. A current limitation of this technology is that the microcarriers are only available in a non-suspendable granular form. The density of these microcarriers further limits the ability to scale-up this technology for large bioreactors, which require a suspendable microbead carrier.
A complementary system using an aragonite (CaCO3) is disclosed in U.S. Pat. No. 5,480,827 (G. Guillemin et al). Although this patent also teaches the importance of calcium in a support system for mammalian cell culture, the calcium compound was not in a suspendable form.
The concept of fabricating a suspendable microcarrier bead with a minor component of glass was discussed by A. Lubiniecki in Large-Scale Mammalian Cell Culture Technology in which a minimal glass coating was applied to a polymer bead substrate by a chemical vapor deposition process or low temperature process. This approach also was disclosed in U.S. Pat. No. 4,448,884 by T. Henderson (see also U.S. Pat. Nos. 4,564,532 and 4,661,407). However, this approach primarily used the polymer bead substrate to maintain suspendability.
An example of the use of non-suspendable or porous ceramic particles for cell culture is taught by U.S. Pat. No. 5,262,320 (G. Stephanopoulos) which describes a packed bed of ceramic particles around and through which oxygen and growth media are circulated to encourage growth of cells. U.S. Pat. No. 4,987,068 (W. Trosch et al.) also teaches the use of porous inorganic (glass) spheres in fixed bed or fluidized bed bioreactors. The pores of the particles act as sites for the culture of cells. Conversely, Richard Peindhl, in U.S. Pat. No. 5,538,887, describes a smooth surface cell culture apparatus which would limit cell attachment to chemical adhesion and prevent mechanical interlocking.
Macroporous glass beads also have been reported by D. Looby and J. Griffiths, xe2x80x9cImobilization of Cells In Porous Carrier Culturexe2x80x9d, Trends in Biotechnology, 8: 204-209, 1990, and magnesium aluminate porous systems by Park and Stephanopolous, xe2x80x9cPacked Bed Reactor With Porous Ceramic Beads for Animal Cell Culture, Biotechnology Bioenginering, 41: 25-34, 1993. Fused alumina fs have been reported by Lee et al, xe2x80x9cHigh Intensity Growth of Adherent Cells On a Porous Ceramic Matrix. Production of Biologicals from Animal Cells in Culture, editors, R. E. et al, Butterworth-Heinemann pp. 400-405, 1991, and polyurethane foam by Matsushita et al, xe2x80x9cHigh Density Culture of Anchorage Dependent Animal Cells by Polyurethane Foam Packed Bed Culture Systemsxe2x80x9d, Applied Microbiology Biotechnology, 35: 159-64, 1991.
Fluidized bed reactors have been used for cell culture as reported by J. M. Davis (editor), Basic Cell Culture, (Cartwright and Shah), Oxford University Press, New York, 1994, but require carrier systems with densities between 1.3 and 1.6 g/cc. According to Cartwright (J. M. Davis, supra.), generally, in fluidized beds, cells do not grow on the exterior surface of carriers where they would be dislodged by inter-particle abrasion. Instead, as with macroporous microcarriers, they colonize the interior pores where they proliferate in a protected microenvironment. As examples, (Cartwright, supra, p. 78) cell carriers used in fluidized beds include glass beads (Siran by Schott Glass), and collagen microspheres produced by Verax. Cartwright also disclosed other conventional microcarriers weighted with TiO2 (Percell Biolytica products) and IAM-carrier polyethylene beads weighted with silica.
Examples of the microcarriers of the present invention are set forth in FIG. 1.
The present invention provides hollow microbeads having a density of about 1.01 grams/cc to about 1.12 grams/cc. More specifically, the hollow microbeads comprise 0 to 100% hydroxylapatite (HA), 0 to 100% tricalcium phosphate (TCP) and/or 0 to 100% other calcium phosphate compounds. The hollow microbeads comprise a wall, wherein the wall may be impermeable to aqueous media. The essentially spherical hollow microbeads can have a diameter of from about 100 micrometers to about 6 millimeters. In another embodiment, the hollow microbead can further comprise a porous coating and/or a biological coating.
The present invention also provides hollow microbeads having a density from about 1.2 grams/cc to 2.0 grams/cc. These hollow microbeads can further comprise a porous coating and/or a biological coating.
Also provided are biomedical implants comprising the above-described microbeads. The biomedical implants can further comprise a biological material or pharmaceutical agent. More specifically, the biomedical implants have a density from about 25% to about 75% of the material""s theoretical density. (By xe2x80x9ctheoretical densityxe2x80x9d is meant the density of a microbead having no pores.) The biomedical implant may comprise a microbead wherein the microbead comprises a wall that is essentially impermeable or porous to aqueous media. The biomedical implant may also comprise microbeads comprising holes, i.e., portals or channels.
Also provided are chromatographic columns comprising the hollow microbeads as set forth above.
Also provided are aggregates comprising the hollow microbeads as set forth above. Such aggregates can be used as biomedical implants and chromatographic columns. The aggregates may be bonded by cementations agents.
The invention further provides hollow and solid glass or polymer microbeads formed with or coated with particulate HA, TCP and other CaPs.
Also provided are hollow and solid microbeads comprising composites of HA, TCP, other CaPs and ceramic, including glass and polymeric materials. These microbeads may have abraded surfaces and aggregates may be made from them. The aggregates may be used in biomedical implants and in chromatographic columns.
Also provided are suspendable and non-suspendable aggregates comprising closed and/or open pores, foamed structures of ceramic, including glass, and/or polymeric composite materials. These aggregates can comprise HA, TCP and other CaP coatings, porous coatings, or biological coatings including growth factors. Biomedical implants can be made comprising these aggregates. Also provided are methods of augmenting tissue comprising implanting these biomedical implants. The biomedical implants may further comprise a biologically active agent and have a density from about 25% to about 75% of the material""s theoretical density. Chromatographic columns also can be made from such aggregates.